Measuring system and method for performing luminometric series analyses as well as multiple cuvette for receiving liquid samples therefor

ABSTRACT

A measuring system and method are provided for performing luminometric series analyses on reaction components to be investigated and the liquid samples containing the magnetizable carrier particles binding the components. Sample chambers receive the liquid samples. These sample chambers are transported to a measuring station on a conveyor. Permanent magnets act on the sample chambers with magnetic fields during transport. A separating station is also provided, which is preferably equipped with a suction and rinsing device to remove the surplus reaction components separated from the carrier particles that accumulate on wall areas of the sample chambers under the influence of the magnetic fields.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of application Ser. No.08/793,090, filed on Jun. 3, 1997, now U.S. Pat. No. 5,985,671 theentire disclosure of which is hereby incorporated by reference.

The invention relates to a measuring system for performing luminometricseries analyses on reaction components to be investigated and the liquidsamples containing the magnetizable carrier particles binding saidcomponents, with a sample chamber that receives the liquid sample andcan be transported to a measuring station on a conveyor, with apermanent magnet that acts on the sample chamber with its magnetic fieldduring transport, as well as a separating station preferably equippedwith a suction and rinsing device to remove the surplus reactioncomponents separated from the carrier particles that accumulate on awall area of the sample chamber under the influence of the magneticfield.

The invention also relates to a method for luminometric series analysesin which a sample chamber, filled with a liquid sample containingreaction components to be investigated and the carrier particles thatare magnetizable and bind the latter, is transported on a conveyor to aluminescence measuring station with the carrier particles beingaccumulated during the course of transport in a collecting area of thesample chamber wall during a separating phase under the influence of amagnetic field and with surplus reaction components being removed fromthe sample chamber in a washing process that follows the separatingphase.

Measuring techniques of this kind serve primarily in medical diagnosis,food-chemistry analysis, biotechnology, and environmental technology forspecific and quantitative determination of very small amounts ofbiomolecules and toxins, with a large number of samples frequentlyhaving to be processed. For measurement, the target substances containedin a sample liquid are labeled in an immunochemical reaction with aspecific antibody bearing a marker (luminogen) capable of luminescence.For concentration with chemical and physical agents, the targetstructure thus obtained is additionally coupled with a magnetic particlecoated with specific antibodies and separated as quantitatively aspossible from the liquid component under the influence of a magneticfield. This component can then be removed by suction or decanting. Themeasuring process itself takes place following the addition of astarting reagent that excites the luminogen and causes it to glow. Thefluorescence photons then emitted are collected by a photodetectordesigned as a photomultiplier which “looks at” the sample volume in adarkened measuring station and produces a recording in the form ofcounting pulses. On the basis of the total photon yield determined inthe form of integrated counting pulses, the concentration of the targetsubstance is finally determined by a calibration relationship.

A device of the species recited at the outset is known (DE 39 26 462 A1)in which a large number of samples is fed in individual test tubes on aconveyor to a separating station. During transport, permanent magnetsare introduced cyclically into the conveyor and carried along with theassociated test tubes through a section of the conveyor. All of thepermanent magnets in this conveyor section are aligned in the same wayand cause the solid magnetic particles to accumulate at a specific pointon the inside wall of each test tube. Although it is possible to usethis device to permit the separating and washing procedures as well asthe subsequent measurement process to take place fully automatically,the entire structure of this system is mechanically complex and requiresa high operating cost due to the handling of a large number ofindividual tubes. In particular, however, it has been found that whenthe magnetic particles accumulate on the walls of the test tubes, theyhave a tendency to clump and to trap free luminogens because of theircoating that bears the antibodies. During luminescence measurement,these luminogens then generate a certain amount of background noise fromwhich the light signals from the target substances can no longer bedistinguished below a resultant limiting concentration for detection.

Therefore the goal of the invention is to improve a measuring system anda method of the species recited at the outset as well as the associatedmultiple cuvette in such fashion that the measurement results arequickly available at a lower detection limit with reduced handlingexpense.

To achieve this goal, the combinations of features included in Claims 1,20, and 26 are proposed. Advantageous embodiments and improvements onthe invention are contained in the dependent claims.

The solution according to the invention is based on the idea ofconnecting several sample chambers with one another as units that can behandled in common and adjusting these in conjunction with the magneticfield configuration in an optimum fashion to match the special nature ofboth the magnetic separating process and the optical measuring process.For this purpose, according to the invention it is proposed that aplurality of cuvette units each forming a sample chamber be connectedtogether to form a multiple cuvette and that at least two permanentmagnets be mounted along the conveyor with a distance between them, withthe permanent magnets penetrating the cuvette units transported pastthem sequentially with their magnetic fields from wall areas that areopposite one another. The action of the magnets placed in stationarypositions on both sides of the conveyor ensures in simple fashion thatthe magnetizable carrier particles that are attracted during cuvettetransport will accumulate alternately, first on one side and then on theother, of the sample chambers, with the free luminogens that adhere tothe carrier particles as they pass through the sample liquidredistributing themselves in the sample liquid.

According to one preferred embodiment of the invention, each permanentmagnet is a double magnet that consists of two bar magnets that arepreferably cylindrical, arranged parallel to one another with oppositepolarity, with the double magnets being rotatable by means of a rotarydrive around an axis of rotation that extends centrally and axiallyparallel, preferably horizontally, and transversely with respect to theconveyor. As a result, a magnetic field is generated that penetrates thecuvette units in the form of a lobe, under whose influence the carrierparticles move along long spiral paths and can accumulate pointwise aspellets on the cuvette walls. The pellets can then be picked up in thepeak area of the elliptical field line with sufficient attractive forceby a double magnet located downstream with a mirror-symmetric action,without carrier particles settling in dead spaces in the individualcuvettes that are poor in field lines.

To generate a magnetic field that is as strong as possible, with a lowdistribution of the scattered field, the two bar magnets are preferablymade of a metal alloy of the rare earths, and coupled magnetically by ayoke at their opposite poles that face away from the conveyor. A strong,highly bundled magnetic field permits rapid and efficient concentrationof the magnetic particles and thus a high sample throughput.

In order to limit the action of the field primarily to the cuvette unitbeing carried past at any given moment, the two bar magnets of eachdouble magnet are rigidly connected together at a distance thatapproximately corresponds to the cross section of the cuvette units.

A pulsed action of permanent magnetic fields on the individual cuvetteunits moving past can be achieved by mounting the double magnets along asection of the conveyor at intervals that correspond to those betweenadjacent cuvette units.

For preconcentration, at the beginning of this section of the conveyor,a nonrotatable double magnet is arranged horizontally, with bar magnetsmounted approximately at intervals equal to those of adjacent cuvetteunits. With its extensive magnetic field, this double magnet penetratesthe entire volume of the sample chamber, thus collecting carrierparticles even from areas that could not be affected by double magnetsdownstream, with their more tightly bundled magnetic fields.

Advantageously, this separation process takes place in successive stepsthrough at least one group of three double magnets arranged side byside, of which the last two in the transport direction are located onthe same side of the conveyor and the first is located on the oppositeside, with a separating station located at the position of the lastdouble magnet. The first two double magnets serve for alternatelyattracting the carrier particles through the sample liquid, while thelast double magnet further compresses the pellet that has alreadyaccumulated and keeps it away from the suction device of the separatingstation.

Another advantageous embodiment of the invention provides that thecuvette units have a lower measuring chamber area and a loading areathat is located above and expands toward a loading opening. As a result,even with small measuring volumes, there is sufficient room for theengagement of a suction and rinsing device that can be lowered fromabove.

It is advantageous for the conditions of the optical measuring processfor the cuvette units to be trapezoidal or triangular in cross sectionand to have a transparent optical measuring window that covers theentire measuring chamber area on one side wall. The large-area measuringwindow increases the outlet area for the luminescence radiation into thehalf-chamber on the detector side, while the sample chamber area thattapers toward the rear, in which the emitted radiation is in any eventmainly reabsorbed in the cloudy suspension of carrier particles, stillhas sufficient cross-sectional area for the intervention of a suctiondevice.

A dimensionally-stable multiple cuvette that can be manufacturedeconomically has, in the form of a one-piece molded plastic part,preferably six cuvette units rigidly connected to one another bylateral, vertical connecting ribs running at the level of the loadingareas and upper horizontal connecting surfaces at the level of theloading openings.

The handling of the multiple cuvettes during their introduction into theconveyor can be facilitated by insertion bevels located at their endsand formed by vertical cross members, each being aligned at an angle tothe connecting surfaces and the connecting ribs on the measuring windowside.

Advantageously, the multiple cuvette can be transported by means of astepping system in a stepwise movement lengthwise along the conveyor. Inthis way it is possible to integrate the transport process in simplefashion into the measurement procedure that is characterized by aplurality of method steps that follow one another in time.

The interference of luminescence radiation between the cuvette units andthe amount of light that passes between them can be reduced by a mattesurface on the multiple cuvette that is roughened except for themeasuring windows of the cuvette units. A further improvement in thisregard can be achieved by the connecting ribs on the measuring windowside and the opposite back of the multiple cuvette each being providedwith an emission window. An additional benefit of the emission window asregards transport function is that the emission windows on the measuringwindow side are made in the form of transport stops that are recessed orproject stepwise on the connecting ribs, said steps engaging a steppingpawl of the stepping system by a ramp, and that the emission window onthe rear is designed as a latching bead for the engagement of a ballunder pressure that secures the position of the multiple cuvette in thestepping system.

To prevent rotation, the multiple cuvette advantageously has centeringrecesses for the engagement of a centering fork, said recessesadvantageously being located eccentrically, formed on the connectingribs, and open at the bottom.

In order to add residual liquid during the suction action of a suctionneedle of the suction device that can be lowered into the cuvette, thecuvette units can have bottoms that slope downward from their measuringwindow sides to the opposite rear sides.

In accordance with the method, the abovementioned goal of the inventionis achieved by virtue of the fact that the carrier particles, during theseparating phase and under the influence of at least two magnetic fieldsthat pass through the sample chamber sequentially and pass throughopposite wall areas, are pulled between two spatially separatecollecting areas through the liquid sample.

Preferably the carrier particles are pulled on spiral paths through theliquid sample under the influence of two rotating magnetic fieldsproduced by two permanent bar magnets with opposite polarity that rotatearound a common central axis, and accumulate in the collecting areas aspellets. Along the long spiral paths, an effective separation of thepreviously accumulated carrier particles takes place together withincreased separation from free surplus luminogens that are not bonded tothe target substance.

The latter can thus be removed from the sample by virtue of the factthat the washing process, in the case of magnetic particles thataccumulate in the form of pellets on the sample chamber wall, isperformed under the continued action of the rotating magnetic field byinjecting and sucking away a rinse fluid. To further increase detectionsensitivity and to suppress the noise caused by free luminogens, aplurality of successive separating phases and washing processes can beprovided in the course of the transport of the sample chambers.

A continuous automatable sample throughput can be made possible byvirtue of the fact that a plurality of sample chambers is transportedsimultaneously on the conveyor in the form of the cuvette units of amultiple cuvette that are connected together and are preferably madetrilateral and prismatic. The multiple cuvettes can then be introducedin succession into the conveyor and can be transported as a connectedtrain of cuvettes lengthwise on the conveyor in a stepped motion.

The invention will now be described in greater detail with reference toan embodiment shown schematically in the drawing.

FIGS. 1 to 3 show a multiple cuvette with six cuvette units in a frontview, top view, and rear view;

FIG. 4 is a representation, enlarged in sections, of a cuvette unit ofthe multiple cuvette in the top view in FIG. 2;

FIG. 5 is a section through the sample chambers of the individualcuvettes along line V—V in FIG. 4;

FIG. 6 is a section along line VI—VI in FIG. 4;

FIG. 7a is a measuring system for performing luminometric seriesanalyses as seen in a top view; and

FIG. 7b shows the measuring system according to FIG. 8a in a view fromthe measuring window side of the cuvette units in their respectivetransport positions on the conveyor; and

FIG. 8 is an enlarged view of a double magnet used in the measuringsystem according to FIG. 7a and made in the form of a horseshoe magnet.

The measuring system 10 shown in FIGS. 7a and b serves to performluminometric series analyses on liquid samples containing targetsubstances to be detected and the labeling substances connectable withthem in an immunochemical detection reaction as well as magnetizablecarrier particles. Liquid samples 12 are transported in a multiplecuvette 14 along a conveyor 16 to a measuring station 18, with permanentmagnets 20-27 designed as double magnets 68 and separating stations 28intended to separate surplus labeling substance acting on multiplecuvettes 14 during transport.

The multiple cuvette 14 shown in FIGS. 1 to 6 is formed as a one-piecemolding from a plastic that is permeable to light and has six connectedcuvette units 30 joined in a row with equal spaces between them. Thecuvette units 30, designed as trilateral prismatic hollow bodies, have alower measuring chamber area 32 and a loading area 36 above that expandsat the top to form a loading opening 34. One side wall 31 of cuvetteunits 30 that are triangular in cross section is designed in measuringchamber area 32 as a surface-polished transparent measuring window 40that extends to cuvette bottom 38, while the other side walls 33, 35have a surface that is rough and matte on the outside. At theirconnecting points, side walls 31, 33, 35 are connected together byinternally concave wall areas. Cuvette bottom 38 is inclined downwardfrom measuring window side 42 to the opposite rear side 44. The rigidconnection of cuvette units 30 to form a multiple cuvette 14 is achievedby connecting ribs 46, 48 on the measuring window side and on the rearthat run at the level of loading areas 36 and by upper connecting areas50 that run at the level of loading openings 34, with multiple cuvette14 being made more rigid by cross members 52 arranged at the ends andcenter. In addition, connecting ribs 46, 48, connecting areas 50, andcross members 52 have a roughened surface structure.

For introduction into conveyor 16, multiple cuvette 14 is provided onits ends with insertion bevels 54, 56 arranged at an angle to connectingareas 50 and connecting ribs 46 on the measuring window side. In orderto permit transport in conveyor 16 by means of a stepping system, notshown, multiple cuvette 14 has on its rear connecting ribs 48 transportstops 58 that project inward stepwise, said stops engaging a steppingpawl of the stepping system by a ramp 60. Hence, the edge 62 that is atthe rear and is not beveled also serves as a transport stop. Inaddition, latching beads 64 are provided on connecting ribs 46 on themeasuring window side opposite transport stops 58 for the engagement ofa ball under pressure that secures the position of multiple cuvette 34in the stepping system. To prevent rotation when multiple cuvette 14 isinserted into conveyor 16, centering recesses 66 located opposite oneanother and open at the bottom are provided in connecting ribs 46, 48 attwo positions that are asymmetric with respect to the arrangement ofcuvette units 30, for the engagement of a centering fork.

As shown in FIG. 8, each double magnet 68 consists of two cylindricalbar magnets 70 with opposite polarity mounted parallel to one another,said magnets being coupled by a yoke 72 to form an arrangement in theshape of a horseshoe magnet. The two bar magnets 70 are kept apart fromone another by a separating layer 74 made of plastic at a distance thatis approximately equal to the width of the measuring windows of cuvetteunits 30. Double magnets 68 formed in this manner are rotatable by meansof a rotary drive 76 around a rotational axis 78 that is centrallyaxially parallel to bar magnets 70, thus producing a rotating magneticfield 80 that extends in the shape of a lobe into the half space locatedin front of their free poles.

According to FIG. 7 the individual rotatable double magnets 21-27 arelocated at stationary positions 3-9 with the spacing of adjacent cuvetteunits 30 in multiple cuvette 14 along conveyor 16 on both sides of thelatter, with rotational axes that are aligned transversely with respectto conveyor 16. At the beginning of conveyor 16 (positions 1 and 2) anonrotatable double magnet 20 is mounted horizontally whose bar magnetsare mounted approximately at the spacing of two adjacent cuvette units30.

Along conveyor 16, a plurality of separating stations 28 equipped withsuction and rinsing devices is mounted above multiple cuvettes 14 beingtransported, said devices being lowerable under program control in suchfashion that they submerge an injection needle 82 for rinsing fluid intoloading area 36 of a cuvette unit 30 located beneath and a suctionneedle 84 into the rear area of cuvette unit 30 down to bottom 38 of thecuvette.

To perform a measurement, the liquid material 12 to be investigatedalong with the target substances to be determined therein, together witha surplus of an antibody against the target substance as well as amarker substance containing luminogens, is added to cuvette units 30. Atthe same time, carrier particles that are magnetizable and made of ironare suspended in material 12 to be investigated, said particles having agrain size of about 10 μm and having their exteriors coated with a latexlayer that also contains specific antibodies against the targetsubstance.

The antibodies of the carrier particles and the labeling substance“recognize” the target molecules to be detected and bind them in animmunochemical reaction. As a result, specific complexes of carrierparticles and molecules of the target and labeling substances areformed. In an incubation process lasting on the order of several minutesto hours, the reaction is brought to equilibrium for the most part atconstant temperature. The actual determination is performed optically,with the luminogens being excited to glow at measuring station 18 andthe yield of luminescence radiation thus produced being measured. Inorder not to distort the measurement results, the free luminogens thatare not bonded to target molecules must be removed from the sample priorto luminescence measurement. For this purpose the substance 12 to beinvestigated is subjected in the manner described below to a separatingprocess that takes place during transport to measuring station 18, saidmethod being performed stepwise corresponding to a stepwise transportmovement of cuvette units 30.

Initially the multiple cuvettes 14 loaded with the samples areintroduced by an automatic handling unit, not shown, of measuring system10 into conveyor 16. Then the cuvette unit 30 which at that moment islocated at input position 1 of conveyor 16 receives additional liquidbuffer (injection needle 86) and the substance to be investigated isexposed for the duration of two transport steps to the extensivemagnetic field of the first static double magnet 20. Under the influenceof magnetic field 88 the iron particles in measuring chamber area 32 arepreconcentrated in measuring chamber area 32 on the inside of themeasuring window side. At the next position (position 3), a rotatingdouble magnet 21 that acts transversely from the same side of conveyor16 compresses the accumulated magnetic particles into a pellet 92 in theform of a dot. Compression is performed by rotating magnetic field 80 insuch fashion that the iron particles are moved on spiral paths into thearea of higher field strengths and, at the center of ellipsoid magneticfield 80, accumulate on the interior of measuring window 40 in acollecting area 90. Then magnetic field 80 of double magnet 68, which interms of its configuration approximately conforms to the inside contourof cuvette unit 30, extends with sufficient attractive force to theopposite rear side 44. The concave wall connections of side walls 31,33, and 35 of the individual cuvettes 30 also prevent carrier particlesfrom settling in corner areas that would otherwise not be reachable bymagnetic field 80. Following this separation, the removal by suction ofthe remaining material to be investigated takes place at the sameposition 3. For this purpose, suction needle 84 is used which can beinserted into rear measuring chamber area 32 while maintaining asufficiently large distance from pellet 92, with pellet 92 adhering tothe side wall because magnetic field 80 is maintained. The nonbondedluminogen is largely removed along with the fluid that is drawn off.Then rinse fluid is added again by means of injection needle 82. At thenext transport position 40, the pellet is picked up in the peak area ofthe magnetic field of the next double magnet 22 that penetrates from theback 44 of cuvette unit 30 and is vorticized spiral-fashion in therinsing liquid so that the free luminogens still contained therein aredissolved again. Following the formation of a pellet 92, in the next twotransport steps 5 and 6, under the influence of double magnets 23, 24that act from measuring window side 42 at the corresponding positions, arenewed vorticization and accumulation of the carrier particles atmeasuring window 40 takes place, followed by another suction andinjection process. The same method steps are repeated a final time atthe next transport positions 7, 8, 9 under the successive influence ofanother group of three double magnets 25, 26, 27 and completed by aresuspension (injection needle 94) of the carrier particles at position10. Then cuvette unit 30 is moved to darkened measuring station 18 wherea luminescence reaction of the luminogens is activated by addingadditional reagents. The luminescence radiation 96 that passes throughmeasuring window 40 and declines in the course of a few seconds isdetected by photodetector 98 and evaluated integrally taking intoaccount calibration relationships and calibration measurements regardingthe concentration of target substance.

In summary, therefore, the following can be determined: the inventionrelates to a measuring system and a method for performing luminometricseries analyses on reaction components to be investigated and on theliquid samples 12 containing magnetizable carrier particles binding saidcomponents. The samples are transported in cuvette units 30 of amultiple cuvette 14 on a conveyor 16 to a measuring station 18 where theconcentration of the target substance to be determined is determined bya luminescence measurement. To remove surplus reaction components thatwould distort measurement, multiple cuvettes 14 are transported past aplurality of rotating permanent magnets 20-27, said magnets, with theirlobe-shaped magnetic fields 80, penetrating cuvette units 30sequentially and from opposite wall areas 40, 44. As a result thecarrier particles are carried along spiral paths through the sampleliquid and concentrated as pellets 92. The surplus reaction componentsseparated from the carrier particles can then be removed in a rinsingand suction process.

What is claimed is:
 1. Multiple cuvette for receiving liquid samples forluminometric series analyses with cuvette units connected together inrows and each forming a sample chamber, with cuvette units (30) having alower measuring chamber area (32) and a loading area (36) located aboveand widening to form a loading opening and being formed to betrapezoidal or triangular in cross section, and having a transparentoptical measuring window (40) on one side wall (31) that coversmeasuring chamber area (32), and with cuvette units (30) being connectedrigidly together by vertical connecting ribs (46, 48) extending at thelevel of the loading areas (36) and by horizontal connecting areas (50)located at the level of the loading openings (34), characterized in thatthe connecting ribs (46, 48) on the measuring window side (42) and theopposite rear side (44) each have an emission window (64, 58) thatprevents light from passing between adjacent cuvette units (30). 2.Multiple cuvette according to claim 1, characterized in that the reademission windows (58) are formed as stepwise recessed or projectingtransport stops on connecting ribs (48), against which stops a ramp (60)abuts.
 3. Multiple cuvette according to claim 1 or 2, characterized inthat it is formed as a molded part from plastic and has four to eight,preferably six cuvette units (30), said units being connected rigidlytogether by lateral vertical connecting ribs (46, 48) running at thelevel of loading areas (36) and by upper connecting areas (50) that arehorizontal and are located at the level of loading openings (34). 4.Multiple cuvette according to claim 3, characterized by insertion bevels(54, 56) at their ends, formed by vertical ribs, each at an angle toconnecting areas (50) and connecting ribs (46) on the measuring windowside.
 5. Multiple cuvette according to one of claims 1 to 3,characterized by a roughened matte surface except for measuring windows(40) of cuvette units (30).
 6. Multiple cuvette according to one ofclaims 1 to 3, characterized in that loading area (36) expands upward toloading opening (34).
 7. Multiple cuvette according to one of claims 1to 3, characterized by eccentrically located centering recesses (66)open at the bottom and formed on connecting ribs (46, 48).
 8. Multiplecuvette according to one of claims 1 to 3, characterized in that cuvetteunits (30) have bottoms (38) that slope downward from their measuringwindow sides (42) to the opposite rear sides (44).
 9. Multiple cuvetteaccording to one of claims 3 to 8, characterized by centering recesses(66) open at the bottom, located eccentrically and formed on connectingribs (46, 48).
 10. Multiple cuvette according to claim 2, 3 or 9,characterized in that cuvette units (30) have bottoms (38) that slopedownward from their measuring window sides (42) to the opposite rearsides (44).